Benzamide derivatives as thrombin inhibitors

ABSTRACT

There are provided according to the invention novel compounds of formula (I)                    
     wherein R 1  represents C 1-4 alkyl or C 3-8 cycloalkyl, R 2  represents C 1-4 alkyl or C 3-4 alkenyl, R 3  represents hydrogen, C 1-3 alkyl or halogen, and R 4  represents C 1-6 alkyl, processes for preparing them, pharmaceutical formulations containing them and their use in therapy particularly as thrombin inhibitors.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a Rule 371 Application of PCT Application No.EP99/07194, filed Sep. 30, 1999, which claims priority to GB ApplicationSerial No. 9821483.6, filed Oct. 3, 1998.

BACKGROUND OF THE INVENTION

This invention relates to a new class of chemical compounds and to theiruse in medicine. In particular, the invention concerns novel amidederivatives, methods for their preparation, pharmaceutical compositionscontaining them and their use as thrombin inhibitors.

Thrombin inhibitors have been described previously in InternationalPatent Application No. WO97/22589.

Thrombin is a serine proteinase present in plasma and is formed byconversion from its prothrombin precursor by the action of Factor Xa.Thrombin plays a central role in the mechanism of blood coagulation byconverting the soluble plasma protein, fibrinogen, into insolublefibrin. The insoluble fibrin matrix is required for the stabilisation ofthe primary hemostatic plug. Many significant disease states are relatedto abnormal hemostasis. With respect to the coronary arterialvasculature, abnormal thrombus formation due to the rupture of anestablished atherosclerotic plaque is the major cause of acutemyocardial infarction and unstable angina. Both treatment of anocclusive coronary thrombus by thrombolytic therapy and percutaneoustransluminal coronary angioplasty (PTCA) are often accompanied by anacute thrombotic reclosure of the affected vessel which requiresimmediate resolution. With respect to the venous vasculature, a highpercentage of patients undergoing major surgery in the lower extremitiesor the abdominal area suffer from thrombus formation in the venousvasculature which can result in reduced blood flow to the affectedextremity and a pre-disposition to pulmonary embolism. Disseminatedintravascular coagulopathy commonly occurs within both vascular systemsduring septic shock, certain viral infections and cancer and ischaracterised by the rapid consumption of coagulation factors andsystemic coagulation which results in the formation of life-threateningthrombi occurring throughout the vasculature leading to widespread organfailure.

Beyond its direct role in the formation of fibrin rich blood clots,thrombin has been reported to have profound bioregulatory effects on anumber of cellular components within the vasculature and blood, (Shuman,M. A., Ann. NY Acad. Sci., 405:349 (1986)).

The inhibition of thrombin has been implicated as a potential treatmentfor a number of disease states. Thrombin inhibitors may be useful in thetreatment of acute vascular diseases such as coronary thrombosis,stroke, pulmonary embolism, deep vein thrombosis, restenosis, atrialfibrillation, myocardial infarction, and unstable angina. They have beendescribed as anti-coagulant agents both in-vivo and ex-vivo, and inoedema and inflammation, whereby a low dose of thrombin inhibitor canreduce platelet and endothelial cell thrombin mediated inflammatoryresponses without concomitant anticoagulant effects. Thrombin has beenreported to contribute to lung fibroblast proliferation, thus, thrombininhibitors could be useful for the treatment of some pulmonary fibroticdiseases. Thrombin inhibitors have also been reported in the treatmentof tumour metastasis whereby the thrombin inhibitor prevents the fibrindeposition and metastasis caused by the inappropriate activation ofFactor X by cysteine proteinases produced by certain tumour cells. Theyhave been shown to inhibit neurite retraction and thus may havepotential in neurogenerative diseases such as Parkinson's andAlzheimer's disease. They have also been reported to be used inconjunction with thrombolytic agents by permitting the use of a lowerdose of thrombolytic agent. Other potential uses have been described inU.S. Pat. No. 5,371,091 for the treatment of Kasabach Merritt Syndromeand haemolytic uremic syndrome, in EP565897 for the prevention of fibrindeposits in the eye during ophthalmic surgery, and in DE4126277 for thetreatment of osteoporosis.

Thus, we have now found a novel class of amide derivatives which act asthrombin inhibitors shown as formula (I)

where

R¹ represents C₁₋₄alkyl or C₃₋₈cycloalkyl;

R² represents C₁₋₄alkyl or C₃₋₄alkenyl;

R³ represents hydrogen, C₁₋₃alkyl or halogen;

R⁴ represents C₁₋₆alkyl;

and pharmaceutically acceptable derivatives or solvates thereof.

Referring to the general formula (I), alkyl includes both straight andbranched chain saturated hydrocarbon groups, e.g. methyl, ethyl andisopropyl; cycloalkyl includes saturated cyclic hydrocarbon groups, e.g.cyclopentyl and cyclohexyl; alkenyl includes both straight and branchedchain hydrocarbon groups containing one double bond, e.g. propenyl,2-methylpropenyl and butenyl.

It will be appreciated that a compound of formula (I) contains a chiralcentre at the position denoted by *. Thus, each compound within formula(I) may exist in two distinct optical isomeric forms. The scope of thepresent invention extends to cover individual enantiomers of compoundsof formula (I) and mixtures of enantiomers of compounds of formula (I)in any proportion, including racemic mixtures. Generally it is preferredto use a compound of formula (I) in the form of a purified singleenantiomer, most preferably the (S) isomer.

Referring to general formula (I), R¹ suitably represents propyl,isopropyl, butyl, cyclopentyl or cyclohexyl. R¹ is preferably isopropyl.

R² is suitably methyl, ethyl, propyl or isopropyl. R² is preferablyethyl.

R³ is suitably methyl or chloro. R³ is preferably methyl.

R⁴ is suitably methyl or ethyl. R⁴ is preferably methyl.

Suitable compounds of general formula (I) for use according to theinvention include:

N-Ethyl-N-isopropyl-3-methyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide;

N,N-Diisopropyl-3-methyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide;

N-Isopropyl-3,N-dimethyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide;

3,N-Dimethyl-N-propyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide;

3-Methyl-N,N-dipropyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide;

N-Ethyl-3-methyl-N-propyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide;

N-Butyl-3-methyl-N-propyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide;

N-Cyclohexyl-N-isopropyl-3-methyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide;

N-Isopropyl-3-methyl-N-propyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide;

3-Chloro-N-isopropyl-N-propyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide;

3-Chloro-N,N-diisopropyl-5-[2-(pyridin-4-ylamino)-butoxy]-benzamide;

and pharmaceutically acceptable derivatives or solvates thereof.

Particular compounds of general formula (I) for use according to theinvention include:

N-Ethyl-N-isopropyl-3-methyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide;and pharmaceutically acceptable derivatives or solvates thereof.

By “a pharmaceutically acceptable derivative” is meant anypharmaceutically acceptable salt, or a metabolically labile derivative,for example a derivative of an amine group, of a compound of formula (I)or any other compound which, upon administration to the recipient, iscapable of providing (directly or indirectly) a compound of formula (I)or an active metabolite or residue thereof. It will be appreciated bythose skilled in the art that the compounds of formula (I) may bemodified to provide pharmaceutically acceptable derivatives thereof atany of the functional groups in the compounds of formula (I). Suchderivatives are clear to those skilled in the art, without undueexperimentation, and with reference to the teaching of Burger'sMedicinal Chemistry And Drug Discovery, 5th Edition, Vol 1: PrinciplesAnd Practice, which is incorporated herein by reference.

Preferred pharmaceutically acceptable derivatives of the compounds offormula (I) are pharmaceutically acceptable salts thereof.

Pharmaceutically acceptable salts of the compounds of formula (I)include those derived from pharmaceutically acceptable inorganic andorganic acids. Examples of suitable acids include hydrochloric,hydrobromic, sulphuric, nitric, perchloric, fumaric, maleic, phosphoric,glycollic, lactic, salicylic, succinic, toluene-p-sulphonic,di-p-toluoyl tartrate, sulfanilic, tartaric, acetic, citric,methanesulphonic, formic, benzoic, malonic, naphthalene-2-sulphonic andbenzenesulphonic acids. Preferred pharmaceutically acceptable salts ofthe compounds of formula (I) include the toluene-p-sulphonic acid salt.Other acids such as oxalic, while not in themselves pharmaceuticallyacceptable may be useful in the preparation of salts useful asintermediates in obtaining compounds of the invention and theirpharmaceutically acceptable acid addition salts.

The suitability of compounds of formula (I) as thrombin inhibitors isexhibited by their ability to inhibit human α-thrombin in a chromogenicassay, using N-p-tosyl-gly-pro-lys p-nitroanilide as the chromogenicsubstrate.

Furthermore, the compounds of formula (I) exhibit effectiveanti-coagulant activity in vitro as indicated by the APTT assays hereindescribed.

Furthermore, the compounds of formula (I) exhibit effectiveanti-thrombotic activity as indicated in the Arterio-Venous Shunt Modelherein described.

Thus, the compounds of formula (I) are useful in the treatment ofclinical conditions susceptible to amelioration by administration of athrombin inhibitor. Such conditions include acute vascular diseases suchas coronary thrombosis, stroke, pulmonary embolism, deep veinthrombosis, peripheral arterial occlusion, restenosis, and atrialfibrillation; in oedema and PAF mediated inflammatory diseases such asadult respiratory shock syndrome, septic shock and reperfusion damage;the treatment of pulmonary fibrosis; the treatment of tumour metastasis;neurogenerative disease such as Parkinson's and Alzheimer's diseases;viral infection; Kasabach Merritt Syndrome; haemolytic uremic syndrome;arthritis; osteoporosis; as anti-coagulants for extracorporeal blood infor example, dialysis, blood filtration, bypass, and blood productstorage; and in the coating of invasive devices such as prostheses,artificial valves and catheters in reducing the risk of thrombusformation.

Accordingly, the present invention provides a method of treatment of amammal, including man, suffering from conditions susceptible toamelioration by a thrombin inhibitor which method comprisesadministering to the subject an effective amount of a compound ofgeneral formula (I) or a pharmaceutically acceptable derivative thereof.

References in this specification to treatment include prophylactictreatment as well as the alleviation of symptoms.

In a further aspect, the present invention provides a compound offormula (I) or a pharmaceutically acceptable derivative thereof for useas a therapeutic agent for use in medicine, particularly human medicine.

In a further aspect, the invention provides the use of a compound ofgeneral formula (I) or a pharmaceutically acceptable derivative thereof,for the manufacture of a medicament for the treatment of a conditionsusceptible to amelioration by a thrombin inhibitor.

While it is possible that, for use in therapy, a compound of theinvention may be administered as the raw chemical it is preferable topresent the active ingredient as a pharmaceutical formulation.

The invention thus further provides a pharmaceutical formulationcomprising a compound of formula (I) or a pharmaceutically acceptablederivative thereof together with one or more pharmaceutically acceptablecarriers therefor and, optionally, other therapeutic and/or prophylacticingredients. The compounds of the present invention may be used incombination with other antithrombotic drugs such as thromboxane receptorantagonists, prostacyclin mimetics, phosphodiesterase inhibitors,fibrinogen antagonists, thrombolytic drugs such as tissue plaminogenactivator and streptokinase, non-steroidal anti-inflammatory drugs suchas aspirin, and the like.

Thus the compounds for use according to the present invention may beformulated for oral, buccal, parenteral, topical, rectal, or transdermaladministration or in a form suitable for administration by inhalation orinsufflation (either through the mouth or the nose).

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents (e.g.pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g. lactose, microcrystalline cellulose orcalcium hydrogen phosphate); lubricants (e.g. magnesium stearate, talcor silica); disintegrants (e.g. potato starch or sodium starchglycollate); or wetting agents (e.g. sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g. lecithin or acacia); non-aqueous vehicles (e.g.almond oil, oily esters, ethyl alcohol or fractionated vegetable oils);and preservatives (e.g. methyl or propyl-p-hydroxybenzoates or sorbicacid). The preparations may also contain buffer salts, flavouring,colouring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound.

For buccal administration the compositions may take the form of tabletsor lozenges formulated in conventional manner.

The compounds according to the present invention may be formulated forparenteral administration by injection e.g. by bolus injection orcontinuous infusion. Formulations for injection may be presented in unitdosage form e.g. in ampoules or in multi-dose containers, with an addedpreservative. The compositions may take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilising and/or dispersingagents. Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g. sterile pyrogen-free water,before use.

The compounds according to the present invention may be formulated fortopical administration by insufflation and inhalation. Examples of typesof preparation for topical administration include sprays and aerosolsfor use in an inhaler or insufflator, or a formulated powder for use inan inhaler.

Powders for external application may be formed with the aid of anysuitable powder base, for example, lactose, talc, or starch. Spraycompositions may be formulated as aqueous solutions or suspensions or asaerosols delivered from pressurised packs, such as metered doseinhalers, with the use of a suitable propellant.

The compounds according to the present invention may also be formulatedin rectal compositions such as suppositories or retention enemas, e.g.containing conventional suppository bases such as cocoa butter or otherglycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously,transcutaneously or intramuscularly) or by intramuscular injection.Thus, for example, the compounds according to the present invention maybe formulated with suitable polymeric or hydrophobic materials (forexample as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt.

A proposed dose of the compounds according to the present invention foradministration to a human (of approximately 70 kg body weight) is 0.1 mgto 1 g, preferably to 1 mg to 500 mg of the active ingredient per unitdose, expressed as the weight of free base. The unit dose may beadministered, for example, 1 to 4 times per day. The dose will depend onthe route of administration. It will be appreciated that it may benecessary to make routine variations to the dosage depending on the ageand weight of the patient as well as the severity of the condition to betreated. The precise dose and route of administration will ultimately beat the discretion of the attendant physician or veterinarian.

The compounds of the invention may be prepared by any of the processesknown in the art for the preparation of similar compounds. For example,according to a first process (A) wherein R¹, R², R³, and R⁴, are aspreviously defined, compounds of formula (I) may be prepared bydeprotection of a compound of formula (II),

where P¹ represents a suitable protecting group such astert-butoxycarbonyl, under suitable conditions, e.g. acidic conditionsfor the removal of a tert-butoxycarbonyl group.

According to a second process, (B), a compound of formula (I) may beprepared by reaction of a compound of formula (III) with a compound offormula (IV)

where R⁷ represents hydrogen, and L represents hydroxyl. The coupling isconveniently carried out using standard reagents such as diethylazodicarboxylate and triphenylphosphine in a suitable solvent such astoluene.

According to a third process, (C), a compound of formula (I) may beprepared by reaction of a compound of formula (III) with a compound offormula (IV) where R⁷ represents hydrogen, and L represents a suitableleaving group, such as chloride, in the presence of a suitable base,such as potassium carbonate. The coupling is conveniently effected in asuitable solvent such as N,N-dimethylformamide, preferably at elevatedtemperature.

According to a fourth process, (D), a compound of formula (I) may beprepared from reaction of compounds of formula (V) and formula (VI),

where R⁷ represents hydrogen. The reaction may be conveniently carriedout in the presence of an activating agent or agents such as1-hydroxybenzotriazole,2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate(TBTU), and a base such as ethyidiisopropylamine in a suitable solventsuch as N,N-dimethylformamide.

A compound of formula (II) may be prepared by reaction of a compound offormula (III) with a compound of formula (IV) where R⁷ represents P¹, asdefined above, and L represents hydroxyl or a suitable leaving groupsuch as 4-toluenesulfonate (tosylate). Where L represents hydroxyl thecoupling is conveniently carried out using conditions as similarly usedfor process (B). Where L represents tosylate the coupling isconveniently carried out in a suitable solvent such asN,N-dimethylformamide in the presence of a suitable base such as sodiumhydride.

A compound of formula (II) may also be prepared by reaction of compoundsof formula (V) and formula (VI) where R⁷ represents P¹ as defined above,suitably using the conditions of process (D).

Compounds of formula (III) may be prepared from compounds of formula(VII)

conveniently using boron tribromide in a suitable solvent such asdichloromethane.

Compounds of formula (III) may also be prepared from compounds offormula (IX)

conveniently by reaction with an acid chloride such as pivaloylchloride, in the presence of a base such as triethylamine, in a suitablesolvent such as toluene, followed by reaction with compounds of formula(VI).

Compounds of formula (VII) may be prepared by reaction of compounds offormula (VII) and formula (VI)

conveniently according to the conditions of process (D). Alternatively,the reaction of compounds of formula (VIII) and formula (VI) may becarried out using oxalyl chloride in the presence ofN,N-dimethylformamide in a suitable solvent such as tetrahydrofuran.

Compounds of formula (V) may be prepared by oxidation of thecorresponding aldehyde of formula (X)

where R⁷ represents hydrogen or P¹. The conversion is effected bytreatment of the aldehyde with a suitable oxidising agent such as sodiumchlorite in the presence of sulfamic acid in a mixture of water and1,4-dioxan.

Compounds of formula (X) may be prepared from compounds of formula (XI)and (IV)

where R⁷ represents hydrogen or P¹ and L represents hydroxyl or asuitable leaving group such as 4-toluenesulfonate (tosylate), providingthat where L is a suitable leaving group, R⁷ preferably represents P¹.Where L represents hydroxyl the coupling is carried out using standardreagents identical to those employed in process (B). Where L representstosylate the coupling is carried in a suitable solvent such asN,N-dimethylformamide in the presence of a suitable base such as sodiumhydride.

Compounds of formula (V) may also be prepared from compounds of formula(XII),

where R⁷ represents hydrogen or P¹ and R⁸ represents a suitableprotecting group such as alkyl, e.g. methyl. The reaction is carried outusing appropriate conditions such as lithium hydroxide in 1,4-dioxan oraqueous sodium hydroxide in ethanol.

Compounds of formula (XII) may be prepared from reaction of compounds offormula (XIII) and (IV)

where R⁷ represents hydrogen or P¹, R⁸ represents a suitable protectinggroup such as alkyl, e.g. methyl, and L represents hydroxyl or asuitable leaving group such as 4-toluenesulfonate (tosylate), providingthat where L is a suitable leaving group, R⁷ preferably represents P¹,using suitable conditions similar to those employed for the synthesis ofcompounds of formula (X).

Compounds of formula (IV), (VI), (VIII), (IX), (XI), and (XIII) areknown in the art or may be prepared by standard methods as hereindescribed.

BIOLOGICAL ASSAYS I. Thrombin Inhibitory Activity

The compounds of the invention possess thrombin inhibitory activity asdetermined in vitro by their ability to inhibit human α-thrombin in achromogenic assay, using N-p-tosyl-gly-pro-lys p-nitroanilide as thechromogenic substrate. All dilutions were made in a buffer consistingof: 50 mM HEPES, 150 mM NaCl, 5 mM CaCl₂ 0.1% PEG and at pH 7.4.Briefly, the substrate (final conc. of 100 μM) was added to thrombin(final conc. of 1 nM) and the reaction monitored for 10 mins at 405 nmusing a Biotek EL340 plate reader; the assay was performed at roomtemperature. To obtain IC₅₀s the data were analyzed using Kineticalc™and processed using ActivityBase™ to obtain the IC₅₀ value. To determinethe IC₅₀ at zero and 15 mins. the compounds were preincubated withthrombin for these times prior to adding the chromogenic substrate.

II. Protocol for the APTT

The compounds of the invention possess anti-coagulant activity asdetermined in vitro by their ability to extend the clotting time ofhuman plasma, the activated partial thromboplastin time (APTT). Pooledcitrated (0.38% trisodium citrate w/v) plasma was prepared from bloodtaken from healthy volunteers and stored at −70° C. The APTT tests wereperformed using a Thrombtrack 4 from Nycomed. Actin reagent (areconstituted extract from dehydrated rabbit brain, also containingellagic acid) was obtained from Baxter Healthcare Corporation USA.Briefly, citrated plasma was added to either compound or distilled waterfollowed by addition of actin reagent. These were then mixed for 2 minat 37° C. before adding calcium chloride to initiate clotting. Compoundsextended the normal clotting time, which is in the range 30-35 seconds,to varying degrees depending on their concentrations. The degrees ofextension of the APTT was calculated by the ratio of clotting times inpresence or absence of compound. The concentration of a compound toextend the ‘normal’ APTT by 1.5× was used as a criterion for comparingthe anti-coagulant activities of compounds.

RESULTS

The results below illustrate the thrombin inhibitory activity and theanti-coagulant activity of a range of compounds of formula (I) using theabove described biological methods:

Example No: IC₅₀ (nM) APTT (nM) 1 <1 40 (1.5x) 2 <1 80 (1.5x) 3 3 90(1.5x) 4 5 97 (1.5x) 5 <1 110 (1.5x)  6 1.5 120 (1.5x)  7 <1 100 (1.31x)8 <1 100 (1.32x) 9 <1 60 (1.5x) 10 3.4 100 (1.28x) 11 26 65 (1.5x)

III. Protocol for Arterio-Venous Shunt Model

The compounds of the invention possess anti-thrombotic activity asdetermined in vivo by their ability to reduce thrombus formation in arat arterio-venous shunt model. Anaesthetised (Inactin 120 mg/kg i.p.)rats were prepared by the insertion of an extracorporeal shunt betweenthe left carotid artery and the right jugular vein. The shunt consistedof two 12 cm lengths of polythene tubing (Portex; 0.58 and 0.86 mminternal diameter respectively) connected by 3 mm (base diameter)silicone rubber bungs (Jencons Scientific Ltd) to a 6 cm length ofpolythene tubing (Portex; 3 mm internal diameter). The tubing wasconnected via drilled holes through the centre of each bung. An 8 cmpiece of silk thread was held taut between the two bungs, passingthrough the central holes, so that it remained longitudinally orientatedin the central portion of the shunt. Before cannulation the shunt wasfilled with 154 mM sodium chloride solution (saline).

Following cannulation a haemostatic clip was left in position on thecarotid artery to prevent blood flow through the shunt. The left carotidartery was also instrumented with an ultrasonic flow probe (TransonicSystems Inc., 0.5 mm) which was connected to a Transonic flow meter(model T206) for the continuous display of phasic carotid artery bloodflow. Continuous carotid artery blood flow was acquired by an MI² dataacquisition system (Modular Instruments Inc.).

Following shunt cannulation, and an equilibration period, the protocolwas commenced by the administration of vehicle or compound. Thepre-treatment time was 30 min and was followed by the removal of thehaemostatic clip from the carotid artery thus allowing blood flowthrough the shunt. Following 15 min of shunt blood flow, the arterialclip was replaced, the shunt removed, and 0.5 ml saline injected slowlythrough the central portion of the shunt to remove free blood. Thecotton thread, and associated thrombus, was carefully removed and theweight of the thrombus determined. Coagulation parameters, includingactivated partial thromboplastin time (APTT), were calculated. A 2 mlblood sample was taken by direct cardiac puncture and transferred to atube containing trisodium citrate (ratio 9:1, final concentration ofcitrate 12.9 mM). The blood sample was mixed gently and transferred toeppendorf tubes and centrifuged at 10000 g for 2 min. The plasma wasdecanted and stored at 4° C. until analysis. All tests were performed ona Sysmex CA5000 automated analyser according to the instruction manual.

Antithrombotic activity was assessed by a decrease in thrombus weight,an extension in the time to occlusion, and an increase in the blood flowarea, and was related to effects upon the coagulation parametersmeasured.

The invention is further illustrated by the following intermediates andexamples.

ABBREVIATIONS

H.p.l.c. high performance liquid chromatography

Rt retention time

DIPEA N-ethyidiisopropylamine

DMF N,N-dimethylformamide

TBTU 2-(1 H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate

DMAP 4-dimethylaminopynidine

br broad

s singlet

d doublet

t triplet

m multiplet

t.l.c. thin layer chromatography

METHODS

Analytical H.p.l.c. was carried out on a Hewlett Packard Series II 1090Liquid Chromatograph using a Rainin Microsorb C18 column (size 4.6×150mm, catalog number 80-215-C5) operating at a flow rate of 1.5 ml/min.Eluents were A: 0.1% trifluoroacetic acid/water, B: 0.05%trifluoroacetic acid/acetonitrile. Gradients:

System 1:15-95% B in A over 15 min

Retention times are given for a wavelength (λ) of 254 nm unlessotherwise stated.

Preparative H.p.l.c.:

System A: Supelcosil LC-ABZ column (size 21.2 mm×25 cm or 21.2 mm×10 cm)operating at 15 ml/min (eluents were A: 0.1% trifluoroacetic acid/water,B: 0.01% trifluoroacetic acid in 95:5 acetonitrile/water).

System B: 50 mm Prochrom column packed with 200 g Sorbsil C60 silica geloperating at 80 mls/min [eluent was:dichloromethane (80), methanol (20),acetic acid (0.5) & ammonia (0.5)].

T.l.c. was carried out using Camiab silica (Polygram® SILG/UV₂₅₄).Eluent was dichloromethane:ethanol:aqueous ammonia in stated ratio.

Flash column chromatography was carried out on Merck silica gel (Merck9385) or using SI Megabond Elut® (normal bonded phase, size 60 cc/10 g)cartridges.

INTERMEDIATE 1 (S)-2-(2,3,5,6-Tetrachloro-pyridin-4-ylamino)-propan-1-ol

To a solution of pentachloropyridine (30 g) in 2-propanol (300 ml) wasadded DIPEA (18 ml), DMAP (0.8 g) and (S)-(+)-2-amino-1-propanol (18 g),and the reaction mixture was heated under reflux for 18 h. Aftercooling, the reaction mixture was concentrated under reduced pressure.The residue was triturated with methanol and filtered to give the titlecompound as a white solid (18 g).

Mass spectrum: Found: MH⁺279.

INTERMEDIATE 2 (S)-2-(Pyridin4-ylamino)-propan-1-ol

A mixture of (S)-2-(2,3,5,6-tetrachloro-pyridin-4-ylamino)-propan-1-ol(6 g), 10% palladium on carbon (3 g), potassium carbonate (14.3 g) andethanol (110 ml) was stirred under an atmosphere of hydrogen for 24 h.The reaction mixture was filtered through Harbolite™ and the filtratewas concentrated under reduced pressure to give the title compound as awhite solid (3.2 g).

Mass spectrum: Found: MH⁺153.

INTERMEDIATE 3 3-Methyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzoic Acid

A mixture of 3-methyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzoic acidmethyl ester (9.5 g) and 2M sodium hydroxide solution (31.5 ml) inethanol (100 ml) was heated at 60° C. for 1 h. On cooling, the reactionmixture was neutralised with 2M hydrochloric acid to pH7 andconcentrated under reduced pressure. The residue was subjected to flashcolumn chromatography, eluting with dichloromethane:ethanol (4:1),ethanol, and methanol:formic acid (10:1), to give an impure sample ofthe title compound. Further purification using megabond flashchromatography, eluting with dichloromethane:methanol (4:1) andmethanol, gave the title compound as a pale yellow solid (6.8 g).

Mass spectrum: Found: MH⁺287.

INTERMEDIATE 4 3-Methyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzoic AcidMethyl Ester

(A): A mixture of methyl 3-hydroxy-5-methylbenzoate¹ (11.2 g),triphenytphosphine (17.7 g), (S)-2-(pyridin-4-ylamino)-propan-1-ol (10.3g) and tetrahydrofuran (300 ml) was treated over 10 min with diethylazodicarboxylate (10.6 ml) and the resultant solution was stirred atambient temperature, under nitrogen for 72 h. The reaction mixture wasconcentrated under reduced pressure and the crude product was subjectedto flash column chromatography, eluting withdichloromethane:ethanol:aqueous ammonia (95:5:0.5), to give the titlecompound as a colourless oil (9.5 g).

Mass spectrum: Found: MH⁺301.

(B): A mixture of methyl 3-hydroxy-5-methylbenzoate¹ (8 g),tributylphosphine (11.9 ml), (S)-2-(pyridin-4-ylamino)-propan-1-ol (4.9g) and toluene (300 ml) was treated with1,1′-(azodicarbonyl)dipiperidine (12.1 g) and the resultant solution wasstirred at ambient temperature, under nitrogen for 18 h. The reactionmixture was filtered and the filtrate concentrated under reducedpressure. The residue was subjected to flash column chromatography,eluting with chloroform:methanol:aqueous ammonia (95:5:1) to give thetitle compound as an oil (13.3 g).

Mass spectrum: Found: MH⁺301.

INTERMEDIATE 5 2-(tert-Butoxycarbonyl-pyridin4-yl-amino)butyric AcidEthyl Ester

To a solution of pyridin-4-yl-carbamic acid tert-butyl ester² (2 g) indry DMF (25 ml) were added sodium hydride (60% dispersion in mineraloil, 0.54 g) and ethyl 2-bromobutyrate (1.7 ml). The mixture was stirredat ambient temperature for 18 h. Water (25 ml) was added, and themixture was extracted with diethyl ether. The combined organic extractswere washed with brine, dried (magnesium sulphate) and concentratedunder reduced pressure. The crude product was subjected to flash columnchromatography, eluting with cyclohexane:ethyl acetate (4:1), to givethe title compound as a colourless oil (0.332 g).

Mass spectrum: Found: MH⁺295.

INTERMEDIATE 6 2-(Pyridin4-yl-amino)-butan-1-ol

A stirred solution of 2-(tert-butoxycarbonyl-pyridin-4-yl-amino)butyricacid ethyl ester (0.33 g) in ethanol (5 ml) was treated with sodiumborohydride (0.12 g) and the stirring was continued for 18 h. Water (1ml) was added and the mixture was concentrated under reduced pressure.The residue was absorbed on to silica and the resulting powder wasloaded on to a flash chromatography column which was eluted withmethanol:chloroform:aqueous ammonia (10:89:1). The title compound (0.168g) was obtained as an oil after evaporation of the product containingfractions.

Mass spectrum: Found: MH⁺167.

INTERMEDIATE 7 3-Choro-N,N-diisopropyl-5-methoxy-benzamide

Oxalyl chloride (2.36 ml) was added dropwise to a solution of DMF (0.1ml) and 3-chloro-5-methoxybenzoic acid³ (4.67 g) in anhydroustetrahydrofuran (100 ml). After 1 h, diisopropylamine (3.75 ml) andDIPEA (9.51 ml) were added and stirring was continued for 18 h. Thereaction mixture was partitioned between ethyl acetate and water, andthe organic layer extracted with 1M hydrochloric acid, saturated aqueoussodium bicarbonate and water. After drying the organic phase with brineand over sodium sulphate, the solvent was removed under reduced pressureto give the title compound as a brown solid (4.6 g).

Mass spectrum: Found: MH⁺270.

INTERMEDIATE 8 3-Chloro-5-hydroxy-N,N-diisopropyl-benzamide

To a stirred solution of 3-chloro-N,N-diisopropyl-5-methoxy-benzamide(2.96 g) in anhydrous dichloromethane (30 ml) at −78° C. was added borontribromide solution in dichloromethane (40 ml). The reaction mixture wasallowed to warm to ambient temperature and stirred for 19 h. Thereaction mixture was cooled to −78° C. and methanol (20 ml) added. Thereaction mixture was allowed to warm back to ambient temperature,stirred for 24 h, and then concentrated under reduced pressure. Theresidue was partitioned between ethyl acetate and saturated aqueoussodium bicarbonate. The organic phase was washed with water and driedwith brine and over sodium sulphate. The crude product was purified byflash column chromatography, eluting with cyclohexane:ethyl acetate(1:1), to give the title compound as a white solid (2.24 g).

H.p.l.c. system 1 Rt 11.1 min.

INTERMEDIATE 9 3-Chloro-5-[2S-(pyridin-4-ylamino)-propoxy]-benzoic AcidMethyl Ester

A mixture of 3-chloro-5-hydroxy-benzoic acid methyl ester⁴ (4.5 g),triphenylphosphine (6.3 g), (S)-2-(pyridin-4-ylamino)-propan-1-ol (3.65g) and tetrahydrofuran (100 ml) was treated over 10 min with diethylazodicarboxylate (5.7 ml) and the resultant solution was stirred atambient temperature, under nitrogen for 96 h. The reaction mixture wasconcentrated under reduced pressure and the crude product was subjectedto flash column chromatography, eluting withdichloromethane:methanol:aqueous ammonia (97:3:0.3) to give the titlecompound as a colourless oil (1.5 g)

Mass spectrum: Found: MH⁺321.

INTERMEDIATE 10 3-Chloro-5-[2S-(pyridin-4-ylamino)-propoxy]-benzoic Acid

3-Chloro-5-[2S-(pyridin-4-ylamino)-propoxy]-benzoic acid methyl ester(1.47 g) in ethanol (15 ml) and 2M sodium hydroxide solution (4.6 ml)were heated at 60° C. for 3 h. The reaction mixture was concentratedunder reduced pressure and acidified with acetic acid to pH4. Theresultant solution was treated with diethyl ether and a solidprecipitated which was filtered off. The solid was stirred in water, andthen filtered to give the title compound as a cream solid (1.1 g)

Mass spectrum: Found: MH⁺307.

INTERMEDIATE 11 N-[(1S)-2-chloro-1-methylethyl]pyridin-4-amineHydrochloride

(S)-2-(Pyridin-4-ylamino)-propan-1-ol (15 g) in dichloromethane (150 ml)was treated with thionyl chloride (59 g) whilst maintaining thetemperature <10° C., and the resultant mixture was stirred at roomtemperature for 18 h. The reaction mixture was concentrated underreduced pressure to give the title compound as a white solid (20.1 g).

Mass spectrum: Found: MH⁺171.

INTERMEDIATE 12 N-Ethyl-3-hydroxy-N-isopropyl-5-methylbenzamide

To a cooled (<50° C.) suspension of 3-hydroxy-5-methylbenzoic acid¹ (50g) in triethylamine (100 g) and toluene (500 ml) under nitrogen, wasadded pivaloyl chloride (97.2 ml), and the resultant mixture was stirredat 0-5° C. for 2 h.

Ethylisopropylamine (55.7 ml) was added, the reaction mixture wasstirred at 0-5° C. for 2 h and allowed to reach room temperature. Themixture was washed twice with water and concentrated under reducedpressure to leave a dark oil. This oil was dissolved in ethanol (500 ml)and treated with 5M NaOH solution (100 g of NaOH in 500 ml of water) for3 h at room temperature. The ethanol was removed under reduced pressureand the resultant basic solution diluted with water and extracted withtoluene. The basic layer was acidified with acetic acid to pH5 and theresultant aqueous mixture extracted with dichloromethane. The combinedorganic extracts were washed with brine and concentrated under reducedpressure to give the title compound as an orange/brown solid (41 g).

Mass spectrum: Found: MH³⁰ 222.

EXAMPLE 1N-Ethyl-N-isopropyl-3-methyl-5-[2S-(pyridin-4-ylamino)-propoxyl-benzamide

To a solution of N-ethyl-3-hydroxy-N-isopropyl-5-methylbenzamide (5 g)in DMF(50 ml) was added potassium carbonate (14.1 g). The mixture washeated to 45° C. and N-[(1S)-2-chloro-1-methylethyl]pyridin-4-aminehydrochloride (9.4 g) was added portionwise over 5 min. The mixture washeated to 115-120° C. and stirred at this temperature for 100 h. Aftercooling to room temperature, water (100 ml) was added and the resultantslurry was extracted with dichloromethane. The combined organic phaseswere washed with 10% NaOH (10 g in 100 ml of water), dried overmagnesium sulphate, filtered and concentrated under reduced pressure togive a brown oil. The oil was the purified by flash columnchromatography, eluting with dichloromethane:methanol:aqueous ammonia(98:1:1)-(94:5:1), to give the title compound as a yellow oil (4.4 g).

Mass spectrum: Found: MH⁺356; ¹H-NMR δ ppm (DMSO-d₆) 8.04(½AA′BB′, 2H),6.90(brs, 1H), 6.72(brs, 1H), 6.67(brs, 1H), 6.58(½AA′BB′, 2H),4.45,3.83(2×brs, 1H), 4.05-3.90(m, 3H), 3.33(m, 2H), 2.32(s, 3H),1.28(d, 3H), 1.30-1.05(m, 9H).

The compound of Example 1 may also be prepared according to thefollowing procedure.

N-Ethyl-N-isopropyl-3-methyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide

A mixture of 3-methyl-5-[2S-(pyridin-4ylamino)-propoxy]-benzoic acid(0.1 g), TBTU (0.225 g), DIPEA (0.5ml) and N-ethylisopropylamine (0.09ml) in dry DMF (2 ml) was stirred at ambient temperature for 60 h. Thereaction was concentrated under reduced pressure and the residuepartitioned between ethyl acetate and saturated aqueous sodiumbicarbonate. The combined organic fractions were dried with brine andover magnesium sulphate, and concentrated under reduced pressure. Thecrude product was subjected to megabond flash column chromatography,eluting with dichloromethane:ethanol:aqueous ammonia (95:5:0.5), to givethe title compound as a yellow oil (0.097 g).

T.l.c. (95:5:0.5) Rf 0.3. Mass spectrum: Found: MH⁺356.

Similarly prepared using commercially available amines, were:

EXAMPLE 2N,N-Diisopropyl-3-methyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide

T.l.c. (95:5:0.5) Rf 0.1. Mass spectrum: Found: MH⁺370.

EXAMPLE 3N-Isopropyl-3,N-dimethyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide

T.l.c. (95:5:0.5) Rf 0.3. Mass spectrum: Found: MH⁺342.

EXAMPLE 43,N-Dimethyl-N-propyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide

T.l.c. (95:5:0.5) Rf 0.3. Mass spectrum: Found: MH⁺342.

EXAMPLE 53-Methyl-N,N-dipropyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide

T.l.c. (95:5:0.5) Rf 0.3. Mass spectrum: Found: MH⁺370.

EXAMPLE 6N-Ethyl-3-methyl-N-propyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide

T.l.c. (95:5:0.5) Rf 0.3. Mass spectrum: Found: MH³⁰ 356.

EXAMPLE 7N-Butyl-3-methyl-N-propyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide

T.l.c. (95:5:0.5) Rf 0.3. Mass spectrum: Found: MH⁺384.

EXAMPLE 8N-Cyclohexyl-N-isopropyl-3-methyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide

T.l.c. (95:5:0.5) Rf 0.3. Mass spectrum: Found: MH⁺410.

EXAMPLE 9N-isopropyl-3-methyl-N-propyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide

T.l.c. (100:8:1) Rf 0.3. Mass spectrum: Found: MH⁺370.

EXAMPLE 103-Chloro-N-isopropyl-N-propyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide

A mixture of 3-chloro-5-[2S-(pyridin-4-ylamino)-propoxy]-benzoic acid(0.1 g), TBTU (0.225 g), DIPEA (0.5 ml) and N-propyl isopropylamine(0.067 g) in dry DMF (2 ml) was stirred at room temperature for 24 h.The mixture was poured into saturated aqueous sodium bicarbonate andextracted with ethyl acetate. The combined organic extracts wereconcentrated under reduced pressure and the residue subjected tomegabond flash column chromatography, eluting withdichloromethane:methanol:aqueous ammonia (95:5:0.5) to give the titlecompound as a yellow oil (0.08 g).

T.l.c. (97:3:0.3) Rf 0.2. Mass spectrum: Found: MH⁺390.

EXAMPLE 113-Chloro-N,N-diisopropyl-5-[2-(pyridin-4-ylamino)-butoxy]-benzamideHydrochloride

A mixture of 3-chloro-5-hydroxy-N,N-diisopropyl-benzamide (0.05 g),2-(pyridin-4-yl-amino)-butan-1-ol (0.023 g), triphenylphosphine (0.04 g)and toluene (1 ml), was treated with diisopropyl azodicarboxylate (0.03ml) and the resultant solution stirred under nitrogen for 8 days. Thereaction mixture was concentrated under reduced pressure and the residuepurified by flash column chromatography, eluting withchloroform:methanol:aqueous ammonia (90:10:1) to give an impure sampleof the title compound. This impure sample was subjected to preparativeH.p.l.c. (system A) and the purified material was treated with 1Mhydrogen chloride in diethyl ether to give the title compound as a gum(0.002 g).

Mass spectrum: Found: MH⁺404; H.p.l.c. system 1 Rt 11.1 min.

EXAMPLE 12N-Ethyl-N-isopropyl-3-methyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamideHydrochloride

N-ethyl-N-isopropyl-3-methyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide(6.21 g) was dissolved in 2M hydrochloric acid (35 ml) and stirred for10 min, and then the mixture was concentrated under reduced pressure.The residue was azeotroped with acetonitrile twice. This overallprocedure was repeated to give the title compound as an amorphous powder(6.19 g).

Mass spectrum: Found: MH⁺356. H.p.l.c. system 1 Rt 7.0 min.

The compound of Example 12 may also be prepared according to thefollowing procedure.

N-Ethyl-N-isopropyl-3-methyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamideHydrochloride

A mixture of 1,1′-(azodicarbonyl)dipiperidine (10.3 g) andtributylphosphine (10.3 g), in toluene (350 ml) was treated with(S)-2-(pyridin-4-ylamino)-propan-1-ol (4.1 g) andN-ethyl-3-hydroxy-N-isopropyl-5-methylbenzamide (12 g) and the resultantsolution was stirred at 40° C., under nitrogen, for 24 h. The reactionmixture was filtered and the filtrate was concentrated under reducedpressure to give a light brown oil which was partially purified bypreparative H.p.l.c. (system B). The resultant oil (21.17 g) wasdissolved in dichloromethane and washed with aqueous ammonia solution,water and brine, and then concentrated under reduced pressure to yieldan oil (5.74 g). This oil was dissolved in tetrahydrofuran (50 ml),added to a mixture of methanol (0.52 ml) and acetyl chloride (1.2 ml)and stirred at room temperature for 30 min. The solution wasconcentrated under reduced pressure, and the residue azeotroped withdiisopropylether to give the title compound as a white foam (3.7 g).

Mass spectrum: Found: MH³⁰ 356; ¹H-NMR δ ppm (DMSO-d₆) 13.62(brs, 1H),8.84(brd, 1H), 8.22, 8.07(brd, 2H), 7.02, 6.93(2×brd, 2H), 6.78(brs,1H), 6.68(brs, 1H), 6.60(brs, 1H), 4.40, 3.76(brs, 1H), 4.22(m, 1H),4.06, 3.98(ABX, 2H), 3.29, 3.12(2×brs, 2H), 2.27(s, 3H), 1.28(d, 3H),1.25-1.00(brm, 9H).

EXAMPLE 13N-ethyl-N-isopropyl-3-methyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide4-methylbenzenesulfonate

To a solution ofN-ethyl-N-isopropyl-3-methyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide(3 g) in isopropylalcohol (30 ml) was added p-toluenesulfonic acid (1.62g) and the resultant solution was stirred at room temperature for 30min. The solvent was removed under reduced pressure to give a paleyellow oil which was redissolved in isopropylalcohol (10 ml). Theresultant solution was then added to diisopropylether (50 ml) whichresulted in the title compound being produced as a white crystallinesolid (3.22 g).

Mass spectrum: Found: MH⁺356; ¹H-NMR δ ppm (DMSO-d₆) 13.09(brs, 1H),8.61(brd, 1H), 8.23, 8.08(2×½AA′BB′, 2H), 7.48(½AA′BB′, 2H),7.11(½AA′BB′, 2H), 7.03,6.88(2×½AA′BB′, 2H), 6.78(brs, 1H), 6.68(brs,1H), 6.60(brs, 1H), 4.40,3.76(2×brs, 1H), 4.22(m, 1H), 4.09,3.97(ABX,2H), 3.29,3.12(2×brs, 2H), 2.28(s, 6H), 1.27(d, 3H), 1.24-1.02(m, 9H).

EXAMPLE 14N-ethyl-N-isopropyl-3-methyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide2-hydroxybenzoate

To a solution ofN-ethyl-N-isopropyl-3-methyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide(0.65 g) in toluene (3.2 ml) was added a solution of salicylic acid(0.25 g) in tetrahydrofuran (1.2 ml) and the resultant solution wasstirred at room temperature for 2 h. The solution was cooled to 0° C.,and diluted with diisopropylether (5 ml); no crystallisation occurred.The solution was then concentrated under reduced pressure to give thetitle compound as a foam (1.0 g)

Mass spectrum: Found: MH⁺356; ¹H-NMR δ ppm (CDCl₃) 8.02(brd, 2H),7.95(dd, 1H), 7.61(brd, 1H), 7.31(dt, 1H), 6.91(brd, 1H), 6.80(dt, 1H),6.70(brs, 1H), 6.66(brd, 2H), 6.56(brs, 1H) 4.59, 3.92(2×brs, 1H),4.07-3.82(3×m, 3H), 3.38, 3.20(2×brs, 2H), 2.30(s, 3H), 1.35(d, 3H),1.26(brt, 3H), 1.13(brd, 6H).

EXAMPLE 15N-ethyl-N-isopropyl-3-methyl-5-[2S-(pyridin-4-ylamino)propoxy]-benzamide(2R,3R)-2,3-dihydroxybutanedioate

To a solution ofN-ethyl-N-isopropyl-3-methyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide(0.65 g) in tetrahydrofuran (3.2 ml) was added a solution of L-tartaricacid (0.28 g) in methanol (5 ml). No crystallisation occurred at roomtemperature. The reaction mixture was concentrated under reducedpressure to give a colourless gum which was dissolved in the minimumquantity of isopropylalcohol and added to excess diisopropylether (250ml). The resultant solution was stirred at room temperature for 2 daysand filtered to give the title compound as a white solid (0.82 g).

Mass spectrum: Found: MH⁺356; ¹H-NMR δ ppm (DMSO-d₆) 8.12(½AA′BB′, 2H),7.97(d, 1H), 6.83(½AA′BB′, 2H), 6.79(brs, 1H), 6.68(brs, 1H), 6.61(brs,1H), 4.40, 3.76(2×brs, 1H), 4.12(m, 1H), 4.04(s+ABX, 3H), 3.98(ABX, 1H),3.28, 3.13(2×brs, 2H), 2.28(s, 3H), 1.27(d, 3H), 1.24-1.00(m, 9H).

EXAMPLE 16N-ethyl-N-isopropyl-3-methyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide4-aminobenzenesulfonate

To a solution ofN-ethyl-N-isopropyl-3-methyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide(0.65 g) in tetrahydrofuran (3.2 ml) was added a slurry of sulphanilicacid (0.32 g) in water (5 ml). The resultant clear solution was stirredat room temperature for 2.5 h and then concentrated under reducedpressure. The residue was dissolved in diisopropylether (30 ml) andafter 2 days filtered to give the title compound as a white powder (0.86g).

Mass spectrum: Found: MH⁺356; ¹H-NMR δ ppm (DMSO-d₆) 13.1(brs, 1H),8.61(brd, 1H), 8.23, 8.08(2×½AA′BB′, 2H), 7.30(½AA′BB′, 2H), 7.03,6.88(2×½AA′BB′, 2H), 6.78(brs, 1H), 6.68(brs, 1H), 6.60(brs, 1H),6.54(½AA′BB′, 2H), 6.02(brs, 2H), 4.40, 3.76(2×brs, 1H), 4.23(m, 1H),4.09, 3.97(ABX, 2H), 3.29, 3.12(2×brs, 2H), 2.28(s, 3H), 1.28(d, 3H),1.24-1.00(m, 9H).

REFERENCES

1) Turner, F A; Gearien, J E, J. Org Chem., 1959, 1952.

2) Kelly, T A; McNeil, D W, Tetrahedron Lett., 1994, 35(48), 9003.

3) Sargent, M V, J. Chem. Soc., Perkin Trans. 1, 1982,1095.

4) Kimio, T; Sumio, S; Masaru, O, Heterocycles, 1985, 23(6), 1483.

Compounds of formula (I) may be included in pharmaceutical formulations,details of such formulations are given below.

TABLETS FOR ORAL ADMINISTRATION

A. Direct Compression % w/w Active ingredient 32.7 Anhydrous lactose36.8 Microcrystalline cellulose 25.0 Pregelatinised maize starch 5.0Magnesium stearate 0.5

The active ingredient was sieved and blended with the excipients. Theresultant mix was compressed into tablets using a tablet machine fittedwith suitable diameter punches.

A rotary machine may also be used for tabletting.

Tablets of various strengths may be prepared by for example altering theratio of active ingredient to lactose or the compression weight andusing punches to suit.

B. Wet Granulation Formulation (i) % w/w Active ingredient 3.5 Lactose73.25 Starch 15.0 Pregelatinised maize starch 7.5 Magnesium stearate0.75

The active ingredient was sieved through a suitable sieve and blendedwith lactose, starch and pregelatinised maize starch. Suitable volumesof purified water were added and the powders were granulated. Afterdrying, the granules were screened and blended with the magnesiumstearate. The granules were then compressed into tablets using suitablediameter punches. The water used for granulation does not appear in thefinal product.

A rotary machine may also be used for tabletting.

Tablets of various strengths may be prepared by for example altering theratio of active ingredient to lactose or the compression weight andusing punches to suit.

Formulation (ii) % w/w Active ingredient/lactose 93.0 granule*Microcrystalline cellulose 5.5 Crosscarmellose sodium 1.0 Magnesiumstearate 0.5 Active ingredient/lactose granule* Active ingredient 50.0Lactose 50.0 Purified water qs + + The water does not appear in thefinal product. Typical range 100-140 g per kg of blend.

The active ingredient and lactose were mixed together and granulated bythe addition of purified water. The granules obtained after mixing weredried and passed through a screen, and the resulting granules were thenmixed with the other tablet core excipients. The mix is compressed intotablets.

A rotary machine may also be used for tabletting.

Tablets of various strengths may be prepared by for example altering theratio of active ingredient to lactose or the compression weight andusing punches to suit.

The tablets may be film coated with suitable film-forming materials suchas hydroxypropyl methylcellulose, preferably incorporating pigments inthe formulation, using standard techniques. Alternatively the tabletsmay be sugar coated, or enteric coated.

Coating Suspension % w/w Hydroxypropyl methylcellulose 10.0 Opaspray 5.0Purified water to 100.0++ or Opadry 10.0 Purified Water to 100.00++ ++The water does not appear in the final product.

COMPRESSION COATED TABLET

The active ingredient may also be formulated as a tablet core usingconventional excipients such as fillers, binders, disintegrants andlubricants, and this core then compressed within an outer tablet(compression coated) using conventional excipients such as apH-independent hydrophilic polymer, fillers, binders, disintegrants andlubricants. This outer coat may also contain active ingredient. Thecompression of both the core and the outer compression coat can beachieved using conventional tabletting machinery.

Such a dosage form can be designed so as to control the release ofactive ingredient as required.

EFFERVESCENT TABLET % w/w Active ingredient 8.75 Sodium bicarbonate41.03 Monosodium citrate anhydrous 41.22 Aspartame 2.5Polyvinylpyrrolidone 2.0 Sodium benzoate 3.0 Orange flavour 1.0 Lemonflavour 0.5 Absolute alcohol for granulation qs

The active ingredient, anhydrous monosodium citrate, sodium bicarbonateand aspartame were mixed together and granulated by the addition of asolution of the polyvinylpyrrolidone in the alcohol. The granulesobtained after mixing were dried and passed through a screen, and theresulting granules were then mixed with the sodium benzoate andflavourings. The granulated material was compressed into tablets usingsuitable diameter punches.

A rotary machine may also be used for tabletting.

LIQUID-FILLED CAPSULE FORMULATIONS FOR ORAL ADMINISTRATION

Liquid formulations were prepared by slow addition of active ingredientinto the other ingredients with constant mixing.

A B % % Example w/w w/w Active ingredient 18.2 18.2 Oleic acid 60.98568.485 Polyethylene glycol 600 7.3 7.3 Propylene glycol 6.0 6.0Polysorbate 80 7.5 — Ascorbyl palmitate 0.015 0.015

The liquid formulations were filled into gelatin capsules, the size ofthe capsule being used and the filler determining the possible fillweight/volume and hence the dose of active ingredient per capsule.

POWDER-FILLED CAPSULES % w/w Active ingredient 24.5 Lactose 75.0Magnesium stearate 0.5

The active ingredient was sieved and blended with the excipients. Themix was filled into hard gelatin capsules using suitable machinery. Thedose is determined by the fill weight and the capsule size.

SYRUP mg/5 ml dose Active ingredient 49.0 Hydroxypropyl methylcellulose22.5 (viscosity type 4000) Buffer qs Flavour qs Colour qs Preservativeqs Sweetener qs Purified water to 5.0 ml

The hydroxypropyl methylcellulose was dispersed in hot water, cooled andthen mixed with an aqueous solution containing the active ingredient andthe other components of the formulation. The resultant solution wasadjusted to volume and mixed. The syrup was clarified by filtration.

SUSPENSION mg/5 ml dose Active ingredient 49.0 Aluminium monostearate75.0 Sweetening agent qs Flavour qs Colour qs Fractionated coconut oilto 5.0 ml

The aluminum monostearate was dispersed in about 90% of the fractionatedcoconut oil. The resulting suspension was heated to 115° C. whilestirring and then cooled. The sweetening agent, flavour and colour wereadded and the active ingredient was suitably dispersed. The suspensionwas made up to volume with the remaining fractionated coconut oil andmixed.

SUB-LINGUAL TABLET % w/w Active ingredient/lactose 49.0 granule*Compressible sugar 50.5 Magnesium stearate 0.5

The active ingredient was sieved through a suitable sieve, blended withthe excipients and compressed using suitable punches. Tablets of variousstrengths may be prepared by altering either the ratio of activeingredient to excipients or the compression weight and using punches tosuit.

A rotary machine may also be used for tabletting.

SUPPOSITORY FOR RECTAL ADMINISTRATION Active ingredient 49.0 mg*Witepsol W32 1.0 g *A proprietary grade of Adeps Solidus Ph Eur

A suspension of the active ingredient in molten Witepsol was preparedand filled using suitable machinery, into 1 g size suppository moulds.

FOR INJECTION % w/v Active ingredient 1.0 Water for injections B.P. to100

Sodium chloride may be added to adjust the tonicity of the solution andthe pH may be adjusted to that of maximum stability and/or to facilitatesoution of the active ingredient using dilute acid or alkali or by theaddittion of suitable buffer salts. Antioxidants and metal chelatingsalts may also be included. The solution is clarified, made up to finalvolume with water and the pH re-measured and adjusted if necessary.

The solution may be packaged for injection, for example by filling andsealing in ampoules, vials or syringes. The ampoules, vials or syringesmay be aseptically filled (e.g. the solution may be sterilised byfiltration and filled into sterile ampoules under aseptic conditions)and/or terminally sterilised (e.g. by heating in an autoclave using oneof the acceptable cycles). The solution may be packed under an inertatmosphere of nitrogen.

Preferably the solution is filled into ampoules, sealed by fusion of theglass and terminally sterilised.

FOR INHALATION Inhalation Cartridges mg/cartridge Active ingredient(micronised) 0.56 Lactose 25.00

The active ingredient was micronised in a fluid energy mill to a fineparticle size range prior to blending with normal tabletting gradelactose in a high energy mixer. The powder blend was filled into No 3hard gelatin capsules on a suitable encapsulating machine. The contentsof the cartridges were administered using a powder inhaler, such as theGlaxo Rotahaler.

Metered Dose Pressurised Aerosol mg/metered Suspension Aerosol dose Percan Active ingredient (micronised) 0.280 73.92 mg Oleic acid 0.020 5.28mg Isopentane 23.64 5.67 g Tetrafluroethane 61.25 14.70 g

The active ingredient was micronised in a fluid energy mill to a fineparticle size range. The oleic acid was mixed with the above at atemperature of 10-15° C. and the micronised drug was mixed into thesolution with a high shear mixer. The suspension was metered intoaluminum aerosol cans and suitable metering valves, delivering 85 mg ofsuspension, were crimped onto the cans and the dichlorodifluoromethanewas pressure filled into the cans through the valves.

NASAL SPRAY % w/v Active ingredient 7.0 Sodium chloride 0.9 Purifiedwater to 100 Shot weight 100 mg (equivalent to 7 mg active ingredient)

The active ingredient and sodium chloride were dissolved in a portion ofthe water, the solution made to volume with the water and the solutionthoroughly mixed.

The pH may be adjusted to facilitate solution of the active ingredient,using acid or alkali and/or subsequently adjusted if necessary takinginto account the pH for optimum stability. Alternatively, suitablebuffer salts may be used. The solution may be preserved with, forexample, benzalkanium chloride and phenylethyl alcohol, for a multi-dosenasal spray.

What is claimed is:
 1. A compound of formula (I)

wherein R¹ represents C₁₋₄alkyl or C₃₋₈cycloalkyl; R² representsC₁₋₄alkyl or C₃₋₄alkenyl; R³ represents hydrogen, C₁₋₃alkyl or halogen;R⁴ represents C₁₋₆alkyl; or a pharmaceutically acceptable derivative orsolvate thereof.
 2. A compound according to claim 1 where R³ representsmethyl, or a pharmaceutically acceptable derivative or solvate thereof.3. A compound according to claim 1 where R⁴ represents methyl, orpharmaceutically acceptable derivative thereof.
 4. A compound selectedfrom:N-Ethyl-N-isopropyl-3-methyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide;N,N-Diisopropyl-3-methyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide;N-Isopropyl-3,N-dimethyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide;3,N-Dimethyl-N-propyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide;3-Methyl-N,N-dipropyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide;N-Ethyl-3-methyl-N-propyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide;N-Butyl-3-methyl-N-propyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide;N-Cyclohexyl-N-isopropyl-3-methyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide;N-Isopropyl-3-methyl-N-propyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide;3-Chloro-N-isopropyl-N-propyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide;3-Chloro-N,N-diisopropyl-5-[2-(pyridin-4-ylamino)-butoxy]-benzamide; ora pharmaceutically acceptable derivative or solvate thereof.
 5. Thecompound:N-ethyl-N-isopropyl-3-methyl-5-[2S-(pyridin-4-ylamino)propoxy]benzamide4-methylbenzenesulfonate, or a pharmaceutically acceptable derivative orsolvate thereof.
 6. A method for the treatment or prophylaxis of acondition susceptible to amelioration by a thrombin inhibitor, in amammal, which method comprises administering to the mammal an effectiveamount of a compound according to claim 1, or a pharmaceuticallyacceptable derivative or solvate thereof.
 7. A pharmaceuticalformulation comprising a compound according to claim 1, or apharmaceutically acceptable derivative or solvate thereof, together withone or more pharmaceutically acceptable carriers therefor.
 8. A processfor preparing a compound of formula (I):

wherein R¹ represents C₁₋₄alkyl or C₃₋₈cycloalkyl; R² representsC₁₋₄alkyl or C₃₋₄alkenyl; R³ represents hydrogen, C₁₋₃alkyl or halogen;R⁴ represents C₁₋₆alkyl; or a pharmaceutically acceptable derivative orsolvate thereof; which process comprises any one of: (A) deprotection ofa compound of formula (II),

where P¹ represents a suitable protecting group; (B) reacting a compoundof formula (III) with a compound of formula (IV)

where R⁷ represents hydrogen, and L represents hydroxyl; (C) reacting acompound of formula (III) with a compound of formula (IV) where R⁷represents hydrogen, and L represents a suitable leaving group, in thepresence of a base; or (D) reacting a compound of formula (V) with acompound of formula (VI),

where R⁷ represents hydrogen, in the presence of an activating agent oragents and a base.
 9. A method for inhibiting thrombin in a subject inneed thereof, said method comprising administering to said subject athrombin inhibiting amount of a compound according to claim
 1. 10. Amethod for the treatment of vascular diseases in a subject, said methodcomprising administering to said subject a thrombin inhibiting amount ofa compound according to claim 1.